The long-term objective of this research is to understand the how Fe-S cluster biogenesis is regulated in vivo. Experiments will be carried out on IscR, a protein that plays a regulatory role in the Isc pathway for Fe- S cluster biogenesis in E. coli. IscR also regulates several other genes encoding for proteins with known or putative roles in Fe-S cluster biogenesis and anaerobic respiratory functions. A fundamental question relating to IscR function is how this protein differentially regulates genes under aerobic and anaerobic conditions, which I propose is due to [2Fe-2S] cluster binding to IscR. Under aerobic conditions, which are known to destabilize some protein-bound Fe-S clusters, I expect that the fraction of cluster-containing IscR is decreased, altering the ability to repress some genes. The specific aim of these experiments is thus to understand the correlation between the [2Fe-2S] cluster occupancy of IscR and the function of this protein in vivo. To test the validity of this hypothesis, I will quantitatively determine the cluster occupancy of IscR under aerobic and anaerobic conditions in vivo using cysteine and iron labeling techniques and isolate mutants of IscR that are altered in their in vivo response to oxygen by performing random mutagenesis. I expect that the cluster occupancy of IscR will be lower under aerobic conditions, and the results should provide insight into the mechanism of IscR regulation of Fe-S cluster biogenesis. This mechanism will be further explored by investigating how cluster occupancy is regulated. In particular, I will determine whether proteins associate with IscR that function to directly influence cluster occupancy using coimmunoprecipitation and co-purification techniques, and I will use genetic techniques to identify new proteins that regulate IscR activity. In addition, I will examine the effects of oxidative and nitrosative stress on the cluster occupancy of IscR. I will also determine whether stress-inducting species act directly to damage the cluster or protein, or if these species act indirectly on IscR by altering the damand for cluster biogenesis. The research described in this proposal will address how IscR mechanistically functions and what role the Fe-S cluster plays in IscR regulation under normal and stressed cellular conditions. How Fe-S cluster biogenesis is regulated is an important question. The generation of Fe-S clusters must be regulated so as to provide proteins with cluster when needed to carry out essential life processes, but also to prevent the overproduction of clusters, which can lead to the accumulation of Fe to toxic levels. In addition, Fe-S clusters can be destroyed by oxidants normally produced by the cell, and these reactions are thought to contribute to cell damage and human disease, including Friedreich's ataxia.